Identification Of Dicer2-Dependent Small RNAs And Regulatory Mechanism Analysis Of Vm-mil RNA6 In Valsa Mali

Apple occupies an important position in Chinese fruit industry,which industry is related to regional economic growth and farmers' poverty alleviation and prosperity.However,apple valsa canker caused by pathogenic fungi V.mali,seriously affects the healthy and sustainable development of apple industry in China.Speeding up the analysis of pathogenic mechanism of the pathogen is of great significance to the formulation of targeted prevention and the effective lasting control of diseases.Recent studies have shown that milRNAs play a key role in the infection process of the pathogen,but the mechanism of post-transcriptional regulation of milRNAs in the interaction between the V.mali and apple still remains uncertain.In the early stage of this study,it was found that the key protein of RNAi pathway exists in V.mali,and play an important role in the pathogenesis of the pathogen.Especially after the deletion of Dicer2,the pathogenicity of V.mali decreased significantly.Therefore,this study used small RNA sequencing technology,degradation group sequencing technology,gene knockout and tobacco co-transformation to analyzed differential sRNAs,identified target genes of milRNAs,and identified the regulatory relationship between milRNA and target genes,and then conducted functional analysis of the two.The results laid an important foundation for comprehensively revealing the pathogenic regulation mechanism of milRNAs upon V.mali.The main results are as follows:1.The sRNA libraries of mycelium of wild-type strain(MVm)and Dicer2 mutant(MD2)of V.mali were constructed.After removing the redundant sequences,the Valid sequences obtained from MVm and MD2 were 102,3889 and 59,3982,respectively.It was found that the deletion of VmDCL2 also had a significant effect on the production of siRNAs,and there were significant differences in the expression pattern of milRNAs between MVm and MD2.Using the degradome deep sequencing technology,we constructed the mycelium(TMVm)and Dicer2 mutant(TMD2)degradome libraries of V.mali.Eleven Dicer2-dependent milRNAs corresponding target genes were screened and analyzed.2.Four positive transformants of Vm-milRNA6(?6-1-22,?6-1-27,?6-2-18 and ?6-2-25)were obtained by knocking out the precursor sequence of Vm-milRNA6.The wild-type strain and the positive transformants were observed and compared,and it was found that the colony morphology were white and nearly round,but the diameter of the positive transformants was significantly reduced and the pathogenicity was significantly enhanced.According to degradome sequencing,function annotation and bioinformatics analysis,VPS10 was identified as a key candidate gene for Vm-milRNA6.The results of co-transformation of Vm-milRNA6 precursor and target gene VPS10 into Nicotiana Benthamiana leaves,showed that obvious green fluorescence was observed on the cell membrane of N.Benthamiana leaf tissue,when injected with VPS10 alone.When Vm-milRNA6 precursor and VPS10 were co-expressed,the green fluorescence was significantly reduced,indicating that Vm-milRNA6 indeed had targeted cutting effect on VPS10.Western Blot also confirmed that the expression level of GFP decreased significantly after co-transformation.This suggests that Vm-milRNA6 is regulated by target gene VPS10.After knocking out VPS10,two positive transformants(?2-2-37 and ?2-2-44)were obtained.Compared with the wild-type strain,the positive transformants strain had the same colony morphology,but the colony diameter was significantly reduced and the pathogenicity was significantly reduced.